The present invention relates to methods for converting Type IIs restriction endonucleases into site specific nicking endonucleases. The engineering theme is based on a naturally existing nicking endonuclease, N.BstNBI, which is related to Type IIs restriction endonucleases. In general, Type IIs endonucleases bind to a specific sequence and cleave both DNA strands near, but not within the specific sequence. The double-stranded cleavage activity of N.BstNBI has been severely limited by natural mutations and thus it nicks only one strand of DNA under standard digestion conditions. In accordance with the present invention, new nicking endonucleases can be engineered from Type IIs endonucleases by either inactivating their second-strand cleavage activity or by swapping the cleavage domains between a target Type IIs enzyme and a known or engineered nicking enzyme.
Restriction endonucleases are enzymes that recognize and cleave specific DNA sequences. Usually there is a corresponding DNA methyltransferase that methylates and therefore protects the endogenous host DNA from digestion by its cognate restriction endonuclease. Restriction endonucleases can be classified into three groups based on cofactor requirements: Type I, II (including IIs), and III.
More than 3000 restriction endonucleases with over two hundred different specificities have been isolated from bacteria (Roberts and Macelis, Nucleic Acids Res. 26:338-350 (1998)). Type II and Type IIs restriction enzymes require only Mg++ as cofactor; both cleave DNA at a specific position, and therefore are useful in genetic engineering and molecular cloning.
Most restriction endonucleases catalyze double-stranded cleavage of DNA substrate via hydrolysis of two phosphodiester bonds on opposite DNA strands (Heitman, Genetic Engineering. 15:57-107 (1993)). For example, Type II enzymes, such as EcoRI and EcoRV, recognize palindromic sequences and cleave both strands symmetrically within the recognition sequence. Type IIs endonucleases recognize asymmetric DNA sequences and cleave both DNA strands outside of the recognition sequence.
There are some proteins in the literature which break only one DNA strand and therefore introduce a nick into the DNA molecule. Most of those proteins are involved in DNA replication, DNA repair, and other DNA-related events (Komberg and Baker, DNA replication. 2nd edit. W.H. Freeman and Company, New York, (1992)). For example, gpII protein of bacteriophage fI recognizes and binds a very complicated sequence at the replication origin of the phage genome. It introduces a nick in the plus strand to initiate rolling circle replication; it is also involved in ligating the displaced plus strand to generate single-stranded circular phage DNA. (Geider et al., J. Biol. Chem. 257:6488-6493 (1982); Higashitani et al., J. Mol. Biol. 237:388-400 (1994)). Another example is the MutH protein, which is involved in DNA mismatch repair in E. coli. MutH binds at dam methylation site (GATC), where it forms a protein complex with nearby MutS which binds to a mismatch.
The MutL protein facilitates this interaction, triggering single-stranded cleavage by MutH at the 5xe2x80x2 end of the unmethylated GATC site. The nick is then translated by an exonuclease to remove the mismatched nucleotide (Modrich, J. Biol. Chem. 264:6597-6600 (1989)).
The nicking enzymes mentioned above are not very useful in the laboratory for manipulating DNA due to the fact that they usually recognize long, complicated sequences and/or are associated with other proteins to form protein complexes which are difficult to manufacture and use. None of these nicking proteins are commercially available. The nicking enzyme N.BstNBI, was found from the thermophilic bacterium Bacillus stearothermophilus (Morgan et al., Biol. Chem. 381:1123-1125 (2000); U.S. Pat. No. 6,191,267). N.BstNBI is an isoschizomer of N.BstSEI (Abdurashitov et al., Mol. Biol. (Mosk) 30:1261-1267 (1996)). Unlike gpII and MutH, N.BstNBI behaves like a restriction endonuclease. It recognizes a simple asymmetric sequence, 5xe2x80x2-GAGTC-3xe2x80x2, and it cleaves only one DNA strand, 4 bases away from the 3xe2x80x2-end of its recognition site, without interaction with other proteins.
Because N.BstNBI acts more like a restriction endonuclease, it should be useful in DNA engineering. For example, it can be used to generate a DNA substrate containing a nick at a specific position. N.BstNBI can also be used to generate DNA with gaps, long overhangs, or other structures. DNA templates containing a nick or gap are useful substrates for researchers in studying DNA replication, DNA repair and other DNA related subjects (Kornberg and Baker, DNA replication. 2nd edit. W.H. Freeman and Company, New York, (1992)). One potential application of the nicking endonuclease is its use in strand displacement amplification (SDA), which is an isothermal DNA amplification technology. SDA provides an alternative to polymerase chain reaction (PCR). It can reach 106-fold amplification in 30 minutes without thermo-cycling. SDA uses a restriction enzyme to nick the DNA and a DNA polymerase to extend the 3xe2x80x2-OH end of the nick and displace the downstream DNA strand (Walker et al., Proc. Natl. Acad. Sci. USA. 89:392-396 (1992)). The SDA assay provides a simple (no temperature cycling, only incubation at 60xc2x0 C.) and very rapid (as short as 15 minutes) detection method and can be used to detect viral or bacterial DNA. SDA is being introduced as a diagnostic method to detect infectious agents, such as Mycobacterium tuberculosis and Chlamydia trachomatis (Walker and Linn, Clin. Chem. 42:1604-1608 (1996); Spears, et al., Anal. Biochem. 247:130-137 (1997)).
For SDA to work, a nick has to be introduced into the DNA template by a restriction enzyme. Most restriction endonucleases make double-stranded cleavages. Therefore, in previous work, substituted xcex1-thio deoxynucleotides (dNTPxcex1S) have been incorporated into the DNA. Many restriction endonucleases will not cleave phosphodiester bonds with (xcex1-thio substitutions. Thus the endonuclease only cleaves the un-substituted linkages which are designed to be within the primer region. The (xcex1-thio deoxynucleotides are eight times more expensive than regular dNTPs (Pharmacia), and are not incorporated well by the Bst DNA polymerase as compared to regular deoxynucleotides (J. Aliotta, L. Higgins, and H. Kong, unpublished observation). Alternatively, if a nicking endonuclease were to be used in SDA, it would introduce a nick into the DNA template naturally. Thus the dNTPxcex1S would no longer be needed for the SDA reaction when a nicking endonuclease is being used. This idea has been tested, and the result agreed with our speculation. The target DNA can be amplified in the presence of the nicking endonuclease N.BstNBI, dNTPs, and Bst DNA polymerase (U.S. Pat. No. 6,191,267).
There is an increasing demand for more nicking endonucleases, because they are useful in SDA and other DNA engineering applications. We have cloned and characterized the nicking endonuclease N.BstNBI and our results show that N.BstNBI is a naturally mutated Type IIs endonuclease with diminished double-stranded cleavage activity (U.S. Pat. No. 6,191,267). The natural occurrence of this type of endonuclease may be quite limited; in any event, assay methods to detect them unambiguously are not available. So far only two nicking endonucleases have been reported and both recognize same specificity (U.S. Pat. No. 6,191,267). The methods disclosed herein provide a novel approach for generating new nicking endonucleases using a protein engineering approach.
Effort has been long taken to engineer novel endonucleases with little success. FokI is a Type IIs restriction enzyme which exhibits a bipartite nature, an N-terminal DNA recognition domain and a C-terminal DNA cleavage domain (Wah et al., Nature 388:97-100 (1997)). The modular nature of FokI led to the invention of several enzymes with new specificities by substituting other DNA binding proteins for the recognition domain. Fusion of the Ubx homeodomain to the FokI cleavage domain yielded an enzyme that cleaves on both sides of the Ubx recognition site (Kim and Chandrasegaran, Proc. Natl. Acad. Sci. U.S.A. 91:883-887 (1994)). Similar approaches have been utilized to create enzymes that can cleave near Z-DNA (Kim et al, Proc. Natl. Acad. Sci. U.S.A. 94:12875-12879 (1997)), and the Gal4 recognition site (Kim et al., Biol. Chem. 379:489495 (1998)). However, two major drawbacks are associated with such chimeric enzymes. First, the chimeric enzymes cleave at multiple sites on both sides of the recognition sequence; therefore, the cleavage specificity is much relaxed. Second, the enzymatic cleavage activity of the chimeric enzymes is very low.
The dimerization interface of FokI is formed by the parallel helices, xcex14 and xcex15, located less than 10 amino acid residues away from its catalytic site of PDxe2x80x94DTK (Wah et al., Proc. Natl. Acad. Sci. USA. 95, 10564-10569 (1998)). Changing D483A and R487A in the xcex14 helix greatly impaired the DNA cleavage activity of FokI (Bitinaite et al., Proc. Natl. Acad. Sci. USA. 95, 10570-10575 (1998)).
In this patent, protein engineering approaches and methods that lead to creation of highly sequence-specific and highly active nicking endonucleases are disclosed. In the first example, a method for engineering a nicking enzyme by disrupting the dimerization domain of the Type IIs endonuclease MlyI is disclosed. In the second example, a method for converting the Type IIs endonucleases AlwI into a nicking enzyme using domain swapping approach is disclosed.
In accordance with the present invention, methods are provided for converting Type IIs restriction endonucleases into nicking endonucleases. In its simplest form, the method comprises identifying a suitable double-stranded nuclease followed by mutation of the dimerization interface responsible for double-stranded cleavage such that the mutated nuclease cleaves only one DNA strand at a specific location within or adjacent the recognition sequence.
In one preferred embodiment, the mutation occurs by substituting one or more amino acid residues required for dimerization/cleavage. In one particularly preferred embodiment illustrating the approach, the Type IIs restriction endonuclease MlyI is mutated by amino acid alteration.
Type IIs restriction endonuclease MlyI recognizes the same GAGTC sequence as N.BstNBI does, but MlyI cleaves both DNA strands 5 bases from the recognition site, while N.BstNBI only cleaves the top strand, 4 bases from the recognition site (FIG. 1A and B). Two amino acid residues (Tyr491 and Lys494) were changed to alanines, which resulted in a nicking endonuclease, N.MlyI. The engineered N.MlyI still recognizes the same GAGTC sequence, but it cleaves only the top strand, 5 bases downstream from GAGTC (FIG. 1C).
In another preferred embodiment, the mutation comprises swapping or substituting the region containing the dimerization interface with one known to be dimerization-defective resulting in cleavage of one, not both, DNA strands. In a particularly preferred embodiment, the dimerization interface of AlwI is replaced by the corresponding domain from N.BstNBI. The Type IIs endonuclease AlwI recognizes GGATC sequence, which is different than the GAGTC sequence recognized by N.BstNBI (FIG. 1D). The dimerization domain of AlwI was replaced by the corresponding domain in N.BstNBI (FIG. 3). The resulting chimeric endonuclease recognizes the same GGATC sequence that AlwI recognizes, but the engineered N.AlwI cleaves on one DNA strand just like the nicking enzyme N.BstNBI (FIG. 1E). Both engineered N.MlyI and N.AlwI are very active, sequence-specific, and strand-specific nicking enzymes.